The relationship between in vitro measures of drug resistance (genotype and phenotype) and in vivo drug activity has only been partially defined. The goal of this project is to better define this relationship using a novel clinical trial design of monitoring viremia after short-term (14-day), single drug discontinuation in patients failing therapy, and relating the change in viremia to 1) in vitro measures of HIV-1 resistance to the specific drug at the time of its discontinuation, and 2) plasma and/or intracellular levels of the active drug. This study will provide a new means of relating in vitro drug resistance and in vivo drug exposure to virologic response. We plan to perform single drug discontinuations with agents from each drug class: nucleoside reverse transcriptase (RT) inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and protease inhibitors (PIs), although our initial efforts have focused on the NRTI and NNRTI classes. Five patients with stable viremia on regimens containing D4T, ddI, or efavirenz have been enrolled in the trial, and have interrupted D4T or efavirenz and continued the remainder of the regimen. Three of 3 patients discontinuing D4T have experienced an increase in viremia, despite 2 of 3 having virus with multiple NRTI mutations associated with cross-resistance to D4T. By contrast, 0 of 2 patients discontinuing efavirenz have had changes in viremia. Additional patients are being enrolled, including those who will discontinue PIs. The information derived from this project should improve our understanding of how best to interpret resistance tests and to use specific antiretroviral drugs in constructing optimal regimens for treatment-experienced patients.